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Cost Action 853
Agricultural Biomarkers for Array Technology
Management Committee Meeting
and combined
Meeting of Working Groups 1 and 3
September 26 - 28, 2002
Swiss Federal Research Station for Fruit-Growing,
Viticulture and Horticulture
Wädenswil, Switzerland
Agenda
Thursday, September 26
13:30 Registration and Welcome
14:00 Meeting
of Working Group 3 "Bio-Informatics and Information
Dissemination"
14:00 - 14:30
Zlatko Trajanoski
Information management systems for functional genomics
14:40 - 15:10 Expert 1: Hubert Charles
ROSO: A software to search optimized oligonucleotidic probes
for microarrays
15:20 - 16:00 Expert 2: Ulrich Wagner
Concepts for biocomputing in a multiuser environment
16:00 - 16:30 Coffee Break
16:30 - 17:00 Jürg E. Frey
Towards the Random Chip - A Biologist's Approach to Bioinformatics
17:10 - 17:30 Patrick DeMarta
Oligonucleotide probe development for diagnostic microarrays
based
on sequence alignments
17:40 - 18:00 Discussion
18:00 End of Working Group 3 Meeting
18:00 - 19:00 Management Committee Meeting,
Part 1 of 3:
1. Opening and welcome by the Chairman
Jürg E. Frey
2. Introduction of new Delegates
3. Approval of the MCM Draft Agenda
4. Approval of the Minutes of the first MCM held in Brussels,
7/8 March 2002
19:00 Closing
20:30 Dinner at the "alti Fabrik"
Friday, September 27
08:30 - 08:40 Meeting of Working Group
1 "Nucleic-Acid based Microarrays"; Peter Bonants
08:40 - 09:00 Cor Schoen
Use of a novel 3D microarray flow through system for plant
pathogen multiplex detection
09:00 - 09:20 Levente Bodrossy
Diagnostic microarrays in microbiology
09:20 - 09:40 José E. Perez-Ortin
Microarray methods for the detection of pathogenic bacteria
and viruses in plants
09:40 - 10:00 Jens Sobek
Towards the random chip: Establishing the practical parameters
for a new versatile microarray
10:00 - 10:20 Coffee Break
10:20 - 10:50 Expert 1: Ralph Schlapbach
Microarraying in the context of functional genomics
10:50 - 11:20 Expert 2: Giuseppe Firrao
Exploring new strategies in loading, attachment and detection
to lower the costs of
DNA array work
11:30 - 13:30 Lunch at the University
of Applied Sciences Wädenswil
13:30 - 14:00 Expert 3: Wolfgang Ludwig
ARB, a graphically oriented softwarepackage comprising various
tools for sequence
database handling and data analysis
14:00 - 14:20 Neil Boonham
Detection of potato viruses using microarrays
14:20 - 14:40 Knut Rudi
Application of sequence-specific labelled DNA probes in
combination with array hybridization for fingerprinting
and microbial community analyses
14:40 - 15:00 Peter Bonants
Multiplex detection of plant pathogens by microarrays: An
innovative tool for plant health management
15:00 - 15:15 Gustavo Nolasco
Typing of Citrus tristeza virus strains by plate hybridization
with panel of probes
15:15 - 15:30 Mogens Nicolaisen
DNA chip diagnostics: related work at Danish Institute of
Agricultural Sciences
15:30 - 15:50 Coffee Break
15:50 - 16:05 Quirico Migheli
Identification of aflatoxin-producing and non-producing
isolates of Aspergillus flavus
and A. parasiticus by RT-PCR
16:05 - 16:20 Miroslav Sip
Parallel detection of potato pathogens: Possibilities and
problems
16:20 - 16:35 Joanna Pulawska
Identification of some economically important pathogens
of fruit trees, shrubs and ornamental plants
16:35 - 16:50 Karel Petrzik
The most important fruit tree viruses in the Czech Republic
16:50 - 17:05 Expert 4: Jana Boben
Exploring Real Time PCR and microarray in plant virus research
17:05 - 17:30 Discussion
17:30 End of Meeting of Working Group
1
17:30 - 18:30 Management Committee Meeting,
Part 2 of 3:
5. Report of the WG-Coordinators on their
activities since our last meeting
a. Nucleic-Acid Based Microarrays, Peter Bonants
b. Protein-Based Microarrays, Ian Barker
c. Bio-Informatics and Information Dissemination, Peter
von Rohr
d. Chip Production and Analysis, Dietmar Blohm
e. Microarray Technology for Environmental Monitoring, Xavier
Nesme
18:30 Closing
Dinner on your own
Saturday, September 28
08:30 - 12:30 Management Committee Meeting, Part 3 of 3
MCM Draft Agenda
6. Information on planned activities for
the next 12 months period
7. Status of the Action
8. Short Term Scientific Missions
9. New research activities
10. Place and date of next meeting
11. Other topics
12:30 End of Management Committee Meeting
of COST Action 853
For those interested:
15:30 - 16:20 Boat trip to Rapperswil
Abstracts
INFORMATION MANAGEMENT SYSTEMS
FOR FUNCTIONAL GENOMICS
Zlatko TRAJANOSKI
Institute of Biomedical Engineering,
Graz University of Technology
Krenngasse 37, 8010 Graz, Austria
Email: zlatko.trajanoski@tugraz.at ; URL http://genome.tugraz.at
While the principles underlying microarray
expression analysis are relatively simple, the challenges
associated with examining expression levels for thousands
of genes in a single experiment, and of presenting those
data in a usable form are substantial. Further, the true
value of the data lies not in the result of any single experiment,
but rather, in examining correlations across multiple experiments.
Developing an accurate interpretation of expression levels
from microarrays requires the development of genomic information
management system capable of effectively capturing the data
as well as tools to make that data accessible to the laboratory
scientist. To that end, we suggest a software development
program aimed at creating a system that makes microarray
expression analysis accessible to a wide range of users.
Primary development is in Java so that, to the greatest
extent possible, the software has cross-platform compatibility.
The components of the system are:
1) Analytical pipeline including modules
for image processing, cluster analysis, promoter analysis,
as well as tools for functional predictions.
2) Data warehouse including tools for storing and retrieving
microarray data.
In order to fully exploit and leverage
the data generated by expression profiling using microarrays,
it is further necessary to integrate expression data with
phenotype, genotype, and information including tissue distribution
and time course expression data gleaned from previous studies.
Currently used tools and methods will be highlighted as
well as future developments and challenges.
ROSO: A SOFTWARE TO SEARCH OPTIMIZED OLIGONUCLEOTIDIC PROBES
FOR MICROARRAYS
Nancie REYMOND1, Hubert CHARLES1, Guillaume BESLON2, Laurent
DURET3 and Jean-Michel FAYARD1.
1 : Laboratory of Functional Biology,
Insects and Interactions (BF2I), UMR INRA / INSA of Lyon,
Villeurbanne , France.
2: Laboratory PRISMa, INSA of Lyon, Villeurbanne, France.
3: Laboratory BBE - UMR CNRS, University Claude Bernard
- Lyon 1, Villeurbanne, France.
Contact: hcharles@insa-lyon.fr
Microarrays are powerful tools for analyzing
and understanding genome functionality. To run microarray
experiments, one needs to calibrate many consecutive steps
in order to avoid variability and to allow experiment comparisons.
In this work, we have developed ROSO ("logiciel de
Recherche et Optimisation de Sondes Oligonucléotidiques"),
a software devoted to design optimal oligonucleotide long
probes (> 30 bp) for microarrays.
ROSO allows users to choose the type of probes, their size
and localization on the gene target, the number of probes
per gene target, as well as different hybridization parameters
such as ion concentrations, melting temperature range and
threshold for secondary structure rejection. ROSO is a web
tool accessible at the following URL (http://pbil.univ-lyon1.fr/roso/loadWkgFiles.php).
Probe optimization process is based on four successive key
steps:
1. Probe specificity. Specificity is calculated
by comparing probes to the overall studied set of genes
(internal genes), but also by comparison with an external
set of genes (any genes user want to avoid any cross-hybridization
with). Specificity is calculated with Blast program. Blast
parameters were estimated to detect a minimal homology of
70 % on 20 nucleotides length.
2. Probe secondary structures (hairpin and homoduplex).
Stability of secondary structures is calculated with the
thermodynamic model of nearest-neighbor.
3. Probe melting temperature (Tm). Tm is calculated with
the thermodynamic model of nearest-neighbor. When at least
one probe is found for each gene, ROSO keeps the set of
optimal probes with the smallest possible Tm variability.
4. Stabilizing criteria. When multiple probes are found
for one target gene, stability criteria (GC rate, GC clamp…)
are calculated and allow to find the best probe. Moreover,
it allows users to calculate Tm of control probes with mismatches.
Different kinds of validation were performed.
First, simulated data have allowed for the comparison with
the reference software Oligo6® and Mfold. ROSO estimations
of Tm and secondary structures were found to be equivalent
or better than Oligo6® and Mfold estimations, for probes
size comprised between 15 and 70 nucleotides. Second, ROSO
was used to design two sets of 541 and 609 probes for specific
bacterial microarrays corresponding to Buchnera aphidicola
and Ralstonia solanacearum. Human and murine probe sets
were also designed.
The work is conducted in collaboration with UMR 5558 (UCB
Lyon) and with support of the Genopôle Rhône-Alpes.
A few other web tools…
Tm calculation
http://www.cbs.dtu.dk/services/DNAarray/probewiz.html
http://www.nwfsc.noaa.gov/protocols/oligoTMcalc.html
http://www.anachem.co.uk/public/new-products/hybsimulator/default.asp
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
http://bioweb.pasteur.fr/seqanal/interfaces/melting.html
Probe and primer design:
http://berry.engin.umich.edu/oligoarray/index.html (JM Rouillard)
http://arrayit.com/Services/ArrayDesign/arraydesign.html
http://ural.wustl.edu/~lif/probe.pl
http://www.labvelocity.com/jellyfish/index.jhtml
Secondary structures:
http://bioinfo.math.rpi.edu/~zukerm/
CONCEPTS FOR BIOCOMPUTING IN A MULTIUSER ENVIRONMENT
Ulrich WAGNER
Functional Genomics Center Zürich,
Uni ETH Zürich, Winterthurerstrasse 190, Y32H52,
CH-8057 Zürich
With the extremely fast development of
the microarray technology, the amount of gene expression
and other functional gene data resulting from microarray
experiments is increasing in a likewise speedy manner. In
spite of the fact that important discoveries have already
been made with the use of microarray experiments, there
is a lack of standards for the representation and even more
importantly for the exchange of microarray data. One of
the reasons for this can be seen in the fact that research
that is carried out making use of microarray experiments
is a relatively new field. Furthermore, gene expression
data are rather complex, as they only make sense when being
seen in connection to the experimental conditions. Thus,
it is has taken some time to gather experience on the levels
of importance of the different aspects of microarray experiments.
In a research environment with a multitude of users, on
the one hand there exists also a multitude of annotation
schemes for the respective data, which are published in
different formats. On the other hand, there exists a natural
interest to exchange data in order to e.g. complement own
research aspects. Therefore, it is highly important to achieve
a general agreement on the minimal amount of information
that is needed in order to correctly interpret the microarray
data and to check the derived results. The Microarray Gene
Expression Data group (MGED) has proposed the document MIAME
(Minimum Information About a Microarray Experiment) to define
this minimum information and to give an answer to the need
of comprehensive annotation necessary to interpret microarray
data. Besides a general agreement on the nature of information
it is also highly important to agree on a definition of
a controlled vocabulary (e.g. by adapting to certain ontologies)
as well as a standardized data format (e.g. by adapting
to XML-derived formats like MAGE-ML) that is needed to efficiently
exchange data and even to create standardized data bases
like the Arrayexpress repository for microarray data designed
by the EBI. So far, the MIAME document has been proposed
as a starting point for a broader community discussion and
hopefully will result in a general agreement.
TOWARDS THE RANDOM CHIP - A BIOLOGIST'S APPROACH TO BIOINFORMATICS
Jürg E. FREY
Swiss Federal Research Station for
Fruit-Growing,, Viticulture and Horticulture (FAW), CH -
8820 Wädenswil, Switzerland
Email: juerg.frey@faw.admin.ch
The microarrays designed to use
for diagnostic purposes are generally based on well described
sequences of nucleic acid. They exploit the variation within
this DNA/RNA-fragment that was previously shown to be low
within a taxon and high between different taxa of interest.
This approach has disadvantages in that it requires developing
new probes for new groups of organisms and that it only
samples information from a small fraction of the genome.
The idea of the random chip is to use many short oligonucleotides
of random sequence and to check the DNA/RNA of each of the
taxa for presence or absence of the corresponding inverse
sequence of each of the short oligonucleotides. The composition
of short oligonucleotides with respect to presence/absence
is different for different taxa and this generates a taxon-specific
pattern on a chip upon hybridisation with labelled primers.
To test if the idea of a random chip can actually work requires
software to query the genomes of several fully sequenced
organisms. Furthermore, to develop a random chip requires
generation of large numbers of short primers and probes
with random DNA sequence that must comply with several requirements
such as identical Tm, no inverse sequences, no hairpin formation
etc. I describe how I addressed these challenges and which
problems remain to be solved.
ALISCAN. AN INTERACTIVE TOOL TO ASSIST THE DESIGN OF SEQUENCE
ALIGNMENT-BASED PROBES
Patrick DEMARTA
Biodiversity s.r.l., Via Corfù,
71, 25100 Brescia
Email: biodiversity@inwind.it, patrick.demarta@libero.it
The use of DNA arrays for diagnostic
purposes introduces problems which were unknown to expression
studies. Probes should be taxon-specific rather than gene-specific.
The presence of within-taxon variability requires that probes
are developed in regions which are conserved within each
taxon in order to provide consistent detection of every
individual.
However, such regions should be variable enough to allow
clear-cut differentiation between the taxa to be discriminated.
Intensive work on sequence alignments is therefore needed
in order to identify the regions which are the most suitable
for probe development.
AliScan is an interactive and flexible tool developed to
assist the design of oligonucleotide probes from sequence
alignment. The user is request to input and alignment in
FASTA format and to arrange sequences into different groups.
The software helps the user to identify the regions in an
alignment which are the most promising for group-specific
probe development, using colour codes to show scores associated
to each nucleotide of the sequence alignment. Scores are
calculated by formulas which could be completely user defined
and which can take into consideration the consensus within
the group, the presence of a given base in other groups,
base ambiguities, probe degeneracy and gaps.
USE OF A NOVEL 3D MICROARRAY FLOW THROUGH SYSTEM
FOR PLANT PATHOGEN MULTIPLEX DETECTION
SCHOEN C*., DE WEERDT M*., HILHORST R#., CHAN A#., BOENDER
P#., ZIJLSTRA C*. and BONANTS P*.
*Division Plant, Wageningen-University;
Plant Research International B.V., P.O. Box 16, NL-6700
AA Wageningen, The Netherlands. E-mail: c.d.schoen@plant.wag-ur.nl
#PamGene International B.V., P.O. Box 1345, NL-5200 BJ Den
Bosch, the Netherlands
Detecting harmful organisms in plant propagation material
is necessary in order to ensure safe and sustainable agriculture.
If different pathogens need to be detected simultaneously,
this approach is difficult and costly.
The newest development in analysis of nucleic acids is the
microarray technology, in which many different oligonucleotides
can be spotted on little more than one square mm. In this
way various target molecules can be detected in the same
sample with increased specificity and meet the demands for
multiplex detection methods of different plant pathogens.
The use of these micro-fabricated DNA analysis tools will
provide the next generation of inexpensive DNA diagnostics
to measure different pathogens in a massively parallel manner
on a single chip.
Recently PamGene B.V. has developed a revolutionary porous
capillary solid phase microarray. This 3D array combines
the unique properties of a two dimensional platform with
the additional benefits of a third dimension, precise fluid
manipulation through the test area. This new platform is
designed to provide rapid, highly sensitive testing capability
for diagnostic research.
The 3D array offers various advantages, including:
1) improved responsiveness and dynamic
range due to the increased surface area relative to a flat
surface geometry;
2) reduced assay times of only 15 minutes instead of 18
hours, due to dynamic fluid delivery to the chip;
3) more uniform probe deposition and higher array densities
due to wetting properties of microporous materials; and
4) Temperature controlled hybridisation followed in real-time,
for each individual oligo spotted on the matrix.
The 3D array has been shown to significantly
improve reaction signals and increase test kinetics making
this assay an extremely fast, sensitive and high performance
microarray alternative to current available platforms. Characteristics
of this hybridisation system will be highlighted.
DIAGNOSTIC MICROARRAYS IN MICROBIOLOGY
Levente BODROSSY, Nancy STRALIS-PAVESE and Angela SESSITSCH
Department of Biotechnology, Division
of Environmental and Life Sciences, Austrian Research Centers,
A-2444 Seibersdorf, Austria, e-mail:levente.bodrossy@arcs.ac.at
The full potential of DNA microarray technology
in high-throughput detection of bacteria and quantitative
assessment of their community structures is widely acknowledged
but not fully realized yet. A generally applicable set of
techniques based on readily available technologies and materials
was developed for the design, production and application
of diagnostic microbial microarrays in molecular ecology,
in clinical, veterinary and plant microbiology as well as
in food quality control. A microarray targeting the particulate
methane monooxygenase (pmoA) and ammonia monooxygenase (amoA)
genes was developed for the detection and quantification
of methanotrophs, nitrifies and functionally related bacteria.
The microarray consists of a set of 59 probes and covers
the whole known diversity of these bacteria and was validated
with a representative set of strains and environmental clones.
Initial tests assessing the quantification potential of
this system showed very good correlation with 26% average
standard deviation from the expected results.
As the cornerstone of diagnostic microbial microarrays,
the design and behaviour of the oligonucleotide probes does
not differ too much between different platforms, most of
the techniques and guidelines presented here will be easily
transferable to emerging novel technologies.
MICROARRAY METHODS FOR THE DETECTION OF PATHOGENIC BACTERIA
AND VIRUSES IN PLANTS
José E. PEREZ-ORTIN
Laboratory of DNA Chips. Universitat
de València. Spain.
Microarrays are capable of analyzing hundreds
of different loci simultaneously in a short period of time.
However, most microchip arrays require large amounts of
template DNA, or RNA, for efficient rapid, passive hybridization.
The oligonucleotide capture probes should be designed to
maintain uniform stringency conditions for each hybridization
reaction. Multiplex amplification is a possible method of
obtaining high concentration of input DNA but often leads
to large decreases in amplification efficiency. Microelectronic
chips are potentially able to circumvent these problems.
Electronically controlled microelectrode arrays are an interesting
option for the DNA analysis as a diagnostic tool. In contrast
to the passive hybridization environment of other methods,
these devices offer the ability to actively transport DNA
to, and to hybridize at, discreet locations on the microelectrode
array surface. We are currently adapting the microelectronic
devices to the plant pathogen detection problem.
A project in collaboration with IVIA (València, Spain)
in being developed on microarrays for detection of viruses
and phtytopathogenic bacteria in potato plants.
TOWARDS THE RANDOM CHIP - ESTABLISHING THE PRACTICAL PARAMETERS
FOR A NEW VERSATILE MICROARRAY
Jens SOBEK
Institute of Medical Radiobiology,
August-Forel-Strasse 7, CH-8008 Zürich, Switzerland
Email: sobek@imr.unizh.ch
A new method is presented that allows
the characterization of genomes based on oligonucleotide
hybridization patterns using microarrays. The method requires
no information on the genome to be characterized. Identification
of organisms on all taxonomic levels will be possible, and
the method also allows to screen for molecular markers for
genetically based characteristics and the identification
of single nucleotide polymorphisms (SNPs).
One of the practical problems is the collective hybridisation
of a large amount of oligonucleotides on the chip. Due to
differences in their melting temperatures it is difficult
to find appropriate hybridisation conditions. Attempts are
presented to obtain proper conditions. Additionally, we
describe a simple method for the production of a highly
versatile polyethylene glycol coated glass slide, the determination
of its properties and its use in hybridisation experiments.
MICROARRAYING IN THE CONTEXT OF FUNCTIONAL GENOMICS
Ralph SCHLAPBACH
Functional Genomics Center Zurich,
Winterthurerstrasse 190, Y 32 H 52, 8057 Zurich, Switzerland
Email: ralph.schlapbach@fgcz.unizh.ch
With the establishment of DNA microarrays
as a research tool for a broad range of questions in vastly
diverse biological systems, researchers are facing large
amounts of complex data generated by this new and important
technology. To avoid the risk of misinterpretation of microarray
data and their significance for the understanding of the
biological system of interest, it is essential to understand
the potential and limitations of microarray data among other
technologies. Using highly parallel and sensitive detection
methods, even today biological questions still have to be
split up according to the complexity of the question, the
molecule classes to be investigated, the practicability
of technologies and the resources needed to implement these
systems on site.
A summary of the main aspects of microarray experiment design,
production setup and data evaluation is put in the context
of a broader range of current high throughput screening
technologies in the areas of genomics, transcriptomics and
proteomics.
EXPLORING NEW STRATEGIES IN LOADING, ATTACHMENT AND DETECTION
TO LOWER THE COSTS OF DNA ARRAY WORK
Giuseppe FIRRAO
Dipartimento di Biologia Applicata
alla Difesa delle Piante
Università di Udine
Although the DNA arrays are regarded as
a promising technology, their application to diagnostics
is severely limited by cost considerations. It is expected
that the cost will be reduced by some recent technical innovations,
but nevertheless the array-based diagnostics may remain
too expensive to reach the agriculture routine diagnostic
lab.
In this talk I will summarise the major reasons of expense
in the conventional array technology and will report the
work carried out in Udine aimed at the definition of a new
low-cost platform. In detail, a method for the parallel
production of medium density DNA arrays to up to 6144 locations
will be presented. According to this method, several individual
flow-through arrays are piled, allowing communications between
channels corresponding to the same location in different
arrays. Each group of corresponding locations of all arrays
is loaded with a single sample delivery of a volume in the
microliter range.
It will be also shown how the use of electrical potential
to link probes to supports and to detect nucleic acids by
Ru(bpy) eletrochemiluminescence can be applied in a cost
effective manner using the technology provided by the printed
circuit board (PCB) industry.
ARB, A GRAPHICALLY ORIENTED SOFTWAREPACKAGE COMPRISING VARIOUS
TOOLS FOR SEQUENCE DATABASE HANDLING AND DATA ANALYSIS
Wolfgang LUDWIG
Department of Microbiology , Technische
Universität München, Am Hochanger 4 , D-85350
Freising, Germany, Email: ludwig@mikro.biologie.tu-muenchen.de
The ARB (arbor, latin, tree) project is
an interdisciplinary bioinformatics initiative of the Lehrstuhl
für Mikrobiologie and the Lehrstuhl für Rechnertechnik
und Rechnerorganisation of the Technical University of Munich.
A comprehensive software package for sequence data as well
as databases for genes and genomes are available at www.arb-home.de.
Initially, ARB was designed for ribosomal RNA data, however,
can also be used for protein data and currently is adapted
for handling genome data.
According to the ARB concept processed sequences and any
other data assigned to the individual sequences are stored
in an hierarchically structured highly compressed central
data base and accessible by various directly cooperating
software tools for visualisation and analysis. Sequence
and other descriptive data can be im- and exported in commonly
used flat file formats. These and any user provided data
are stored in individual data base fields linked to the
respective sequence or accessible in external databases
via internet connection. Flexible search tools can be used
for visualisation, selection, and extraction of the database
entries. Automated aligners and powerful primary and secondary
structure editors allow processing and visualisation of
the sequences as well as sequence derived profiles and filters.
The most commonly applied treeing approaches for phylogenetic
analyses are implemented. A special version of a maximum
parsimony based algorithm allows to reconstruct, and evaluate
big trees (more than 30.000 sequences) as well as phylogenetic
analyses of heterogenous data sets (i.e. full and partial
sequences). The trees can be used for database structuring
as well as data access and visualisation. Any user defined
selection of database field entries can be visualised at
the terminal nodes of a tree. The ARB_PT-server (positional
tree) allows rapid identification of sequence similarities
or peculiarities. It is used by tools for finding the next
relatives, automated alignment as well as design and evaluation
of specific probes. The probe tools search for diagnostic
sequence stretches for user defined organisms or phylogenetic
groups. The potential target sites are further evaluated
according to criteria of (hybridisation) technical relevance.
Tools for the automated design and evaluation of comprehensive
probe sets for micro arrays according to the multiple probe
concept as well as for the interpretation of chip hybridisation
patterns are currently under development.
Further ongoing software development concerns ARB-Genome
which allows the comparative analysis of annotated genomes
and - according to the ARB integrated database concept -
the combination of the sequences with experimental data
from differential cultivation, expression and proteome studies.
Processed ARB databases are maintained for evolutionary
conserved genes or gene products such as rRNAs, ribosomal
proteins, elongation factors, ATPase subunits, RNA polymerases,
DNA Gyrases, heat shock proteins, recA, aminoacyl tRNA sythetases.
DETECTION OF POTATO VIRUSES USING MICROARRAYS
Neil BOONHAM1, Kathy WALSH1, Kathryn MADAGAN2 and Ian BARKER1
1Central Science Laboratory, Sand
Hutton, York, YO41 1LZ, UK.
2University of York, York UK.
Currently most diagnostic methodology is geared towards
detection of a very specific target species. In order to
test for a long list of unknowns a number of assays need
to be run in parallel to reach a single result. The methods
that are available for virus testing using a generic approach,
namely electron microscopy, test plant inoculations and
more recently MALDI TOF all give an identification to the
genus level, follow up testing using another method is needed
to get identification to the species level. The microarray
method described in this paper addresses this problem by
presenting a technology that can be used to test for a large
number of targets using a completely generic technology.
In the context of virus detection, the issues of specificity
and sensitivity have been examined. The method has been
shown to be able to discriminate sequences with less than
80% sequence identity, and hence useful for discriminating
at the species level, but with broad specificity being able
to detect sequences with greater than 90% sequence identity,
thus the method should be able to cope well with the intrinsic
variability found within the genomes of RNA viruses. The
sensitivity of the assay was found to be comparable with
ELISA.
APPLICATION OF SEQUENCE-SPECIFIC LABELED DNA PROBES IN COMBINATION
WITH ARRAY HYBRIDIZATION FOR FINGERPRINTING AND MICROBIAL
COMMUNITY ANALYSES
Knut RUDI
MATFORSK Norwegian Food Research
Institute, Ås, Norway
Analyses of complex microbial communities
are becoming increasingly important. Bottlenecks in these
analyses, however, are the tools to actually describe the
biodiversity. Novel approaches for a DNA array based analyzes
of microbial communities are presented. In these approaches,
the specificity obtained by sequence-specific labelling
of DNA probes is combined with the possibility of detecting
several different probes simultaneously by DNA array hybridization.
Examples from both fingerprinting and 16S rDNA community
analyses will be given.
MULTIPLEX DETECTION OF PLANT PATHOGENS BY MICROARRAYS: AN
INNOVATIVE TOOL FOR PLANT HEALTH MANAGEMENT
BONANTS P1*., DE WEERDT M1., VAN BECKHOVEN J1., HILHORST
R2., CHAN A2., BOENDER P2., ZIJLSTRA C1. and SCHOEN C1.
1Plant Research International B.V.,
P.O. Box 16, NL-6700 AA Wageningen, The Netherlands.
*E-mail: p.j.m.bonants@plant.wag-ur.nl
2PamGene International B.V., P.O. Box 1345, NL-5200 BJ Den
Bosch, the Netherlands.
Detecting harmful organisms in plant propagation
material is necessary in order to ensure safe and sustainable
agriculture. If different pathogens need to be detected
simultaneously, this approach is costly. The multiplicity
of assays available for a specific pathogen leads to a lack
of consistency among the various testing agencies in Europe
and hampers standardisation.
The newest development in analysis of nucleic acids is the
microarray technology, in which different oligos can be
spotted on little more than one square mm. The use of these
micro-fabricated DNA analysis tools will provide the next
generation of inexpensive DNA diagnostics to measure different
pathogens in a massively parallel manner on a single chip.
To develop the microarray technology for diagnostic purposes
generic DNA/RNA extraction and generic pre-amplification
methods to increase sensitivity have to be developed.
Recently PamGene B.V. has developed a revolutionary porous
capillary solid phase microarray. The capacity of this 3D-array
to bind oligonucleotides is higher than that of a 2D-glass
array resulting in a higher sensitivity. Moreover, the porous
solid phase allows flow through measurements, resulting
in fast hybridisation times of only 15 minutes instead of
18 hours as on glass. Using a temperature control system
hybridisation can be adjusted instantaneously and together
with the use of real-time monitoring, hybridisation kinetics
and melting temperature can be determined for each individual
oligo spotted on the matrix. By using different probes per
pathogen the specificity can be increased even further.
Generic amplification of different targets coupled to array
detection makes this system a useful tool in multiplex detection
of plant pathogens. Recent data to detect different plant-pathogens
(viruses, bacteria, fungi and nematodes) in this multiplex
setting will be discussed.
TYPING OF CITRUS TRISTEZA VIRUS STRAINS BY PLATE HYBRIDIZATION
WITH A PANEL OF PROBES
Gustavo NOLASCO
Universidade do Algarve - FERN, Campus
de Gambelas, 8000 Faro, Portugal
Citrus tristeza virus causes the most
economically damaging viral disease of citrus. Natural infections
are caused by diverse strains whose economic effects may
be very different. In the last years important efforts have
been made to develop tools for quick typing of strains.
Sequence data from about 150 coat protein gene accessions
obtained in the Genbank, University of Florida and our Laboratory
was aligned and 7 groups of strains were defined based on
nucleotide diversity. Interestingly, the groups so defined
depicted a good relationship with the symptomathology produced.
Parsimonious informative sites were located in each group
and a panel of probe sequences designed in such way that
the sequences of each group had a characteristic signature
of reaction with the panel of probes. These probes have
been used as capture probes in asymmetric PCR ELISA assays
for typing isolates from different origins. In these assays
the rate of substrate hydrolysis is measured and, by comparison
with the reaction of typical strains of each group, the
mixture of strains composing each isolate is determined.
Besides epidemiological studies, from a practical point
of view, this provides a very convenient way to detect isolates
which may hide severe strains.
DNA CHIP DIAGNOSTICS: RELATED WORK AT DANISH INSTITUTE OF
AGRICULTURAL SCIENCES
Mogens NICOLAISEN
Danish Institute of Agricultural
Sciences
Dept. of Plant Protection
Research Centre Flakkebjerg
A short overview of initiatives on diagnostic
DNA chips in Denmark will be given. Work in our lab on the
genomic characterization of plant pathogens (virus and fungi)
providing a contribution to the basis of DNA chips will
be presented. A project on the expression profiling and
detection of mycotoxin producing Fusarium will be described.
IDENTIFICATION OF AFLATOXIN-PRODUCING AND NON-PRODUCING
ISOLATES OF ASPERGILLUS FLAVUS AND A. PARASITICUS BY REVERSE
TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)
Barbara SCHERM1, Michele PALOMBA2,Virgilio BALMAS1, and
Quirico MIGHELI1*
Department of Plant Protection -
Center for Biotechnology Development and Biodiversity Research,
University of Sassari, Via De Nicola 9, I-07100 Sassari,
Italy,1 and Dipartimento Farmaco-Chimico-Tossicologico,
University of Sassari, Via Muroni 23, I-07100 Sassari, Italy2
Aflatoxins are polyketide secondary metabolites
that are produced by foodstuff- and animal feed-contaminating
members in the Aspergillus section Flavi, particularly A.
flavus, A. parasiticus and A. nomius. Due to the toxic and
carcinogenic properties of aflatoxins, there is a need to
develop rapid and sensitive methods to detect the presence
of aflatoxigenic Aspergilli in contaminated foods and feeds.
However, not all Aspergillus strains are able to produce
aflatoxins, and this prompted the adoption of multiple screening
techniques to ascertain the real toxigenic potential of
contaminating molds.
Conventional methods used to distinguish among toxigenic
and non-toxigenic isolates in the A. flavus group involve
culturing the fungus in suitable inducing media, extracting
aflatoxin with organic solvents and monitoring their presence
by chromatographic techniques.
To meet the need for methodologies which may be applicable
to screen large numbers of strains in a reasonable time,
alternative methods were developed. These are either based
on the use of complex media to detect the natural fluorescence
of aflatoxins released by the growing mycelium or rely on
multiplex PCR detection of genes involved in the aflatoxin
biosynthetic pathway.
The generally accepted pathway for aflatoxin biosynthesis
involves over 20 enzymatic reactions, and most of the corresponding
genes have been now isolated and characterised. Recent studies
suggest that regulation of aflatoxin biosynthesis in Aspergillus
spp. involves a complex pattern of positive- and negative-acting
transcriptional regulatory factors, which are affected by
physiological response to both external and internal stimuli.
Therefore, while rapid and accurate, the screening methods
based on PCR detection of key genes in the biosynthetic
pathway of aflatoxins may fail to distinguish true aflatoxigenic
isolates from the complex of Aspergillus spp. contaminating
food and feed.
Our aim was to test the reliability of the RT-PCR technique
in differentiating between aflatoxigenic and non-aflatoxigenic
isolates of the A. flavus group. In the present study, new
sets of primers matching a series of key genes in the aflatoxin
biosynthetic pathway were designed and used in RT-PCR experiments
to distinguish, among a collection of well-characterized
isolates of A. flavus and A. parasiticus, those potentially
dangerous by correlating the expression of genes involved
in aflatoxin biosynthesis and the production of aflatoxins
in inducing and non-inducing media measured by high-performance
liquid chromatography (HPLC)/mass spectrometry. The transcription
of some key genes was correlated to the production of aflatoxin
determined by HPLC/mass spectrometry. These results open
the perspective to adopt RT-PCR as well as other cDNA-based
micro- and macroarray techniques to rapidly identify toxigenic
isolates of Aspergillus spp.
PARALLEL DETECTION OF POTATO PATHOGENS: POSSIBILITIES AND
PROBLEMS
Miroslav ŠÍPa,b
a Institute of Plant Molecular Biology,
Academy of Sciences of the Czech Republic,
bUniversity of South Bohemia, Faculty of Health and Social
Sciences, Branišovská 31, 370 05 Ceské
Budejovice, Czech Republic, e-mail: sip@umbr.cas.cz
Parallel detection of potato viruses is
planned using the new microarray technology. This approach
consists in anchoring an array of virus-specific sequences
on a glass plate and subsequent hybridization with labelled
samples of nucleic acids from the tested plants.
The importance of potato as nutrition source makes a reliable
screening of its viral pathogens an economical necessity.
In our project, we focus on the seven most widely distributed
potato viruses: PVY and PVA potyviruses, PVX potexvirus
and PVS and PVM carlaviruses, potato leafroll luteovirus
and the newly detected potato mop-top potamovirus.
To achieve this goal, viral RNA was isolated from infected
indicator plants. Primers for amplification of conservative
as well as variable regions of the virus genomes were prepared
and RT - PCR was performed. 400 - 600 bp DNA amplicons were
cloned to pBSK(+) vector. The corresponding E. coli clones
are kept in the LB medium containing 15% of glycerole.
The clones will be used for design of a DNA-chip. Selected
sequences will be immobilized on a glass plate and used
for parallel detection of the above-mentioned viruses.
In the present phase of the development extensive testing
of selected sequences is being performed to achieve high
selectivity and to avoid false positive results.
IDENTIFICATION OF SOME ECONOMICALLY IMPORTANT PATHOGENS
OF FRUIT TREES, SHRUBS AND ORNAMENTAL PLANTS
Joanna PULAWSKA
Research Institute of Pomology and
Floriculture, Skierniewice, Poland
Diverse research on molecular detection
of plant pathogenic viruses, bacteria and fungi is conducted
in Research Institute of Pomology and Floriculture. One
of the purpose of these studies is to develop DNA markers
suitable for detection of bacterial plant pathogens: tumorigenic
Agrobacterium - crown gall agent and Erwinia amylovora -
?fire blight agent. Another purpose is to discover DNA -
based system for identification strains belonging to different
taxons of genus Agrobacterium. In case of fungal pathogens,
attention is directed to three Phytophthora species: P.
citricola, P. cryptogea, P. cinnamomi and P. ramorum, which
are devastating pathogens for ornamental nurseries.
A set of markers will be designed to recognize and identify
the isolates collected in the Polish nurseries - in microbiological
cultures, infected plant material and substrates using probes
existing in literature. The aim of the research project
on viruses and virus-like pathogens is to provide DNA probes
suitable for their detection.
The work is concentrated on following viruses and phytoplasmas:
plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV),
apple stem pitting virus (ASPV), blackcurrant reversion
virus (BRV), apple chlorotic leaf spot virus (ACSLV), apple
stem grooving virus (ASGV), prune dwarf virus (PDV), pear
decline MLO (PD-MLO). Most of potential probes are already
available as cloned cDNAs. All molecular markers are predicted
as probes for microarray technology.
THE MOST IMPORTANT FRUIT TREE VIRUSES IN THE CZECH REPUBLIC
(APPLE MOSAIC, PRUNE DWARF, PRUNUS NECROTIC RINGSPOT ILARVIRUSES
& PLUM POX POTYVIRUS)
Karel PETRZIK
Institute of Plant Molecular Biology,
Academy of Sciences of the Czech Republic
Nucleotide sequence analysis of the capsid
protein genes of the four viruses revealed regions of high
and low variability suitable for specific and/or non specific
primer design. Apple mosaic ilarvirus (24 sequences) revealed
three highly variable regions between nt 70 and 290. There
still exists 35nt long region after nt 414 with two variable
position only. Quite similar features posses the PNRSV,
where the most variable region was located between nt 120
and 300 and the distal part of the gene was the most conservative.
The movement protein-CP intergenic region of PNRSV is also
highly variable and copy the phylogenetic relationships
of the CP gene. In PDV (15 sequences) there was not so distinct
variable hotspot in the proximal part.
This is the only ilarvirus, where conservative sequence
longer than 20 nt was found (23nt after nt 470). The RNA4
subgenomic promoter sequence was successfully PCR tested
for PDV isolate-independent amplification. The variability
of the plum pox virus (56 analysed sequences) CP reflects
the known secondary arrangement of the protein: highly variable
proximal part but several more-than 20nt long stretches
in the distal part of the gene.
EXPLORING REAL TIME PCR AND MICROARRAY IN PLANT VIRUS RESEARCH
Jana BOBEN, Maja RAVNIKAR, Nataša PETROVIÈ
National Institute of Biology, Department
of Plant Physiology and Biotechnology,
Veèna pot 111, SI-1000 Ljubljana, Slovenia
The research work of the Department
of Plant Physiology and Biotechnology focuses mainly on
studies of the plant - pathogen interactions. Lately, Real
Time PCR and microarray have also been incorporated into
different research projects.
Microarray technique was used in physiological studies of
defence response in potato to potato virus Y, NTN strain
(PVYNTN). Libraries of induced and repressed genes of potato
in response to the infection with PVYNTN were prepared,
using a subtraction hybridisation method. Microarrays were
printed and used to detect differences in gene expression
following the infection, and to identify the differences
in response to viral infection among potato cultivars with
different susceptibility to PVYNTN. Currently, the gene
expression kinetics of selected genes in different Solanum
tuberosum cultivars following the PVYNTN infection is studied,
using Real Time PCR method.
We have been using Real Time PCR method for detection of
genetically modified organisms (GMOs) to distinguish the
virus from the transgene by combine detection of virus CaMV
promoter and coat protein (Cp). In research of plant-virus
interaction, Real Time PCR was used to detect the level
of PVYNTN accumulation in sensitive and resistant potato
cultivars and in determination of PVYNTN coat protein expression
in transgenic potato plants.
Currently, the work is focused on a model virus, a Tomato
mosaic virus (ToMV), where primers and hybridisation probe
were designed for Real Time PCR quantification in applying
a new chromatographic method using monolith disks for virus
concentration. Real Time PCR is also planned to be explored
for detection of economically important plant pathogenic
viruses which occur in plants in very low concentrations,
which are hard to detect by conventional methods, such as
Rupestris Stem Pitting associated Virus 1 (RSPaV-1 ).
List of Participants
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